At-line coupling of LC–MS to bioaffinity and selectivity assessment for metabolic profiling of ligands towards chemokine receptors CXCR1 and CXCR2

• A method for metabolic profiling of ligands towards the CXCR1 and CXCR2 is reported.
• Bioaffinity and selectivity are assessed after at-line nanofractionation of mixtures.
• Accurate MS and MS/MS measurements allowed structure elucidation of metabolites.

This study describes an analytical method for bioaffinity and selectivity assessment of CXCR2 antagonists and their metabolites. The method is based on liquid chromatographic separation (LC) of metabolic mixtures followed by parallel mass spectrometry (MS) identification and bioaffinity determination. The bioaffinity is assessed using radioligand binding assays in 96-well plates after at-line nanofractionation.The described method was optimized for chemokines and low-molecular weight CXCR2 ligands. The limits of detection (LODs; injected amounts) for MK-7123, a high affinity binder to both CXCR1 and CXCR2 receptors belonging to the diaminocyclobutendione chemical class, were 40 pmol in CXCR1 binding and 8 pmol in CXCR2 binding. For CXCL8, the LOD was 5 pmol in both binding assays. A control compound was always taken along with each bioassay plate as triplicate dose-response curve. For MK-7123, the calculated IC50 values were 314 ± 59 nM (CXCR1 binding) and 38 ± 11 nM (CXCR2 binding). For CXCL8, the IC50 values were 6.9 ± 1.4 nM (CXCR1 binding) and 2.7 ± 1.3 nM (CXCR2 binding). After optimization, the method was applied to the analysis of metabolic mixtures of eight LMW CXCR2 antagonists generated by incubation with pig liver microsomes. Moreover, metabolic profiling of the MK-7123 compound was described using the developed method. Three bioactive metabolites were found, two of which were (partially) identified. This method is suitable for bioaffinity and selectivity assessment of mixtures targeting the CXCR2. In contrary to conventional LC–MS based metabolic profiling studies done at the early lead discovery stage, additional qualitative bioactivity information of drug metabolites is obtained with the method described.